It is thought that osteoblasts follow a maturation pathway similar to that of hematopoietic cells, arising from mesenchymal stem cells and passing through a series of discrete phenotypic stages that result in mature osteoblasts which may further mature into osteocytes. Limited information is known about the pathway of osteoblast maturation, particularly at the early stages prior to the appearance of mature osteoblasts, because of the inability to isolate, maintain and characterize cells at these early stages. Preliminary data from the principal investigator indicate that a distinct subpopulation of cells expressing osteoprogenitor (OP) -like characteristics can be isolated from murine long bone; the long-term goal of this proposal is to isolate and characterize the OP from adult mouse long bone and to understand the cellular and molecular events of their differentiation. One encompassing Specific Aim will be pursued: to identify, clone and characterize the OP present in bone. Relative to this: (A) a method to isolate OP cells from adult murine long bones in a reproducible manner will be verified. To determine whether the cells are OPs, their phenotype will be characterized by: (1) assessing their capacity for proliferation, self renewal; (2) alkaline phosphatase activity; (3) synthesis of bone matrix proteins; (4) mineralization in vitro; (5) cyclic AMP production in response to parathyroid hormone; (6) expression of cell surface differentiation antigens; and (7) secretion of, and regulation by osteotropic factors. Regulation of these responses by BMP, dexamethasone, or 1,25(OH)2D3 will also be determined; (B) the cells will be cloned and then the clones will be characterized for the OP phenotype; (C) appropriate clones will be used to raise monoclonal antibodies specific for OP cells; and (D) in vivo activity will be determined by: (1) implantation to measure their ability to produce bone; (2) tracking the cells to analyze their homing specificity; and (3) measuring the change in OP number during fracture repair. Isolation of such cells would provide a source for detailed characterization of osteoblast differentiation, which is poorly understood at present, and may provide the foundation for the emergence of a new therapeutic technology to treat a variety of bone abnormalities including age-related osteopenia.